A number of synthetic basal media have been developed for animal cell culture, especially for mammalian and insect cells. Commercially available media include: Eagle media, M 199, Dulbecco's modified Eagle's medium (DME), Ham's F10 and F12, RPM1 1640, Grace's Insect media and Leibovitz L15. These media, in conjunction with bovine serum or specific growth factors, are often sufficient to support animal cell growth. The formulation of these media, however, are insufficient for sustained growth and proliferation of marine invertebrate cells.
Historically, there has been no flexible basal nutrient media available which can support the in vitro proliferation or preservation of cells from a diverse number of multicellular, eukaryotic, marine invertebrate species.
Recent advances in cellular biology have demonstrated the importance of the synergistic involvement of a variety of media components, including ions, nutrients, growth factors and attachment matrices in the development and proliferation of higher eukaryotic cells.
The inorganic salts in culture media have two major functions. First, salt concentrations are created to approach the natural salt concentration levels in the environment from which the cells are derived. This will minimize any deviation in osmotic pressure on the cells that would require energy consuming ionic pumps to maintain cellular integrity.
Secondly, these salts include many ions which are utilized as enzymatic cofactors and intracellular messengers. Often, for ions to be effectively transported into the cell, additional carrier molecules are needed in the medium. In mammalian systems, transferrin is a carrier of iron. In marine invertebrate systems, it has been demonstrated that metal chelators like diethylenetetramine pentaacetic acid ("DTPA"), citrate and amino acids (lysine and taurine) help to transport metal ions into molluscan tissue. See Coombs, T. C. (1977) Proc. Anal. Div. Chem. Soc. 14:219.
Although most mammalian culture media utilize glucose as the main carbon source, others have demonstrated the benefits of alternative carbon sources for invertebrate culture. See Grace, T. D. C. (1962) Nature 195:788-789, and Leibovitz, A. (1983) Am J. Hyg. 78:173-180.
It is known that many animal cells grow best when attached to natural substrates like collagen, laminin, and fibronectin. It has been shown that the addition of ascorbic acid to culture medium increased the production and deposition of collagen by mammalian cells. See Engvall, E., et al (1986) J. Cell Biol. 102:703-710. The production of collagen by cells in culture helps to create a natural matrix for growth. This type of matrix is very important for many marine invertebrate cells. Sponges, for example, are mostly cells on a collagen mesh.
In a general basal media it is very difficult to define all the requirements animal cells need for growth. The use of bovine or fetal bovine serum as a supplement, as used in mammalian culture systems, does not work with marine invertebrate cells. Serum or hemolymph from marine gastropods, such as Haliotis sp. and Strombus sp., offer an alternative and effective source of additional lipids, trace metals, growth factors and nutrients. These improve the cultured growth of marine invertebrates.
The preservation of cultured cells and a method for preserving and shipping field samples of marine invertebrates offer both economical and environmental savings by reducing or eliminating large scale re-collections of organisms of interest. Serman, J. K. (1964) Proc. Soc. Exptl. Biol. Med. 117:251-264 discloses the value of adding dimethyl sulfoxide (DMSO) to medium in order to prevent ice crystals from rupturing cells during freezing.
Accordingly, it is an object of the present invention to provide a system which employs a basal nutrient medium which is designed to facilitate the in vitro growth, proliferation, and cryopreservation of multicellular, marine invertebrate cells.
It is another object of the present invention to provide a system that will accommodate a wide variety of species and cell types, rather than optimize for any single specific cell line.
It is a further object of the present invention to provide a system employing media that is reliable, convenient to use, and cost effective in its manufacture.